Apolipoprotein (apo) Al is the main structural component of plasma HDL. Plasma levels of HDL and apoAl are inversely related to CHD rates, and their manipulation is a therapeutic intervention for atherosclerosis. ApoAl is the driver of reverse cholesterol transport (RCT), the pathway through which excess peripheral cholesterol is delivered to the hepatocyte. The mechanism through which RCT promotes vascular health is not understood, and the possibility exists that apoAl and HDL exert direct protective effects in the artery wall. In this light, higher HDL levels might simply be related to higher concentrations of HDL in the artery wall, which may be the true effector of plaque stabilization. In the previous cycle of this grant we have shown that the exclusive production of apoAl from arterial macrophages leads to high concentrations of apoAl in the plaque, with minimal amounts of apoAl in plasma and no changes in plasma HDL. This intervention resulted in the complete prevention of atherosclerosis in mouse models with different levels of plasma HDL, ranging from almost none to twice normal. We have also shown that arterial expression of apoAl significantly reduces the atherogenesis induced by apoE deficiency, suggesting that macrophage apoAl can counter heavy lipid burden. We have accomplished this through a combined approach of transgenic, macrophage-specific apoAl expression, and bone marrow cell transduction using apoAI-expressing retroviral vectors. We are now proposing the natural progression of these investigative lines, aiming for mechanistic discoveries, technical innovation, and clinical translatability. We will develop lentiviral vectors expressing apoAl constructs driven by the physiologic ME2 promoter, which is sensitive to cholesterol loading through the physiologic LXR loop. In this system, macrophage-specific expression of apoAl will be up-regulated during the transformation of the macrophage into a foam cell. We will also produce transgenic mice that express apoAl driven by the same cholesterol-sensitive promoter. Another technological advance will be the use of purified hematopoietic stem cells (HSC) and sub-lethal doses of radiation. Using these tools, we will evaluate the effects of human apoAl and single amino-acid variants on vascular lipid accumulation and inflammation in vivo and in vitro. The natural apoAl variants are known to lower HDL levels with diverging effects on atherogenesis. The main hypotheses to be tested are: 1) The vascular effects of apoAl can be dissociated from plasma HDL levels; 2) ApoAl has direct effects on macrophage cholesterol homeostasis, inflammation, and oxidation. These studies have the potential to uncover the basis for the therapeutic effect of HDL on the arterial plaque. [unreadable] [unreadable] [unreadable] [unreadable]